The skin is a potential route for delivery of pharmaceutical or cosmetically active agents to the body. However, the skin is not generally thought of as an efficient delivery route, due to the low permeability of the stratum corneum and the epidermis in general. Traditionally, topical application of pharmaceutical therapeutic agents has been targeted at localized dermatological sites. More recently, transdermal techniques have been used for systemic targeting especially as this route bypasses the hepatic circulation where degradation of the active agent may occur.
Ultrasound can be used to deliver molecules to within the skin. When ultrasound is used in this context it is termed “sonophoresis”. Ultrasound applied to the skin has two main effects. First, cavitation results from the rapidly oscillating pressure field, causing bubble formation and collapse, which mechanically creates channels through the stratum corneum. The second effect is the direct heating of the material through which the sound waves are travelling, due to attenuation of the acoustic energy through reflection, absorption and dispersion. In skin, this occurs up to four times more than other tissues due to its heterogeneity. Heating is known to disrupt the lipid bilayer system in the stratum corneum also contributing to the enhanced permeability of the epidermis. Several factors can affect the heating capacity of ultrasound, including:
(i) applying ultrasound in continuous rather than pulsed mode,
(ii) prolonging the exposure time,
(iii) focusing the ultrasound rather than using unfocused application,
(iv) avoidance of using aqueous gels which are used to decrease the degree of reflection,
(v) applying the ultrasound at higher power densities,
(vi) application of ultrasound to tissues immediately adjacent to bone.
With ultrasound, diffusion of low molecular weight molecules has been shown to increase by 2-5000 times across isolated epidermis in vitro and by up to 1700 times in theoretical studies. Even large molecule drugs such as insulin and heparin have been delivered effectively when using 15 minutes of 20 kHz US. One in vitro study found that poly-L-lysine molecules of up to 51 kDa could be delivered with ultrasound at 20 kHz and intensities in the range of 2 to 50 W/cm2. By way of explaining this increase in permeation, some studies have reported an increase in the number of pores rather an increase in the individual pore diameters (28±12 Å). However, the term ‘sonomacroporation’ has been adopted for specific ultrasound that actually causes larger pore formation.
The permeability of the skin is increased by disruption of the intercellular lipids through heating and/or mechanical stress, and through the increase in porosity. Temperature rises of 6° C. (1 MHz, 0.25 W/cm2) to 50° C. (20 kHz, 10-30 W/cm2) have been reported, but rises as little as 11° C. (1 MHz, 2 W/cm2) have been shown to cause skin damage. Continuous mode ultrasound at an intensity of 1 W/cm2 raises the temperature of tissue at a depth of 3 cm to 40° C. in 10 minutes. For smaller molecules, such as mannitol, enhancement of permeation through the skin occurs when ultrasound is applied as a pre-treatment or simultaneously with application of the molecule; whereas for large molecules such as insulin, enhancement of permeation has only been recorded during application of ultrasound.
Ultrasound can be used to improve transdermal drug delivery. WO 99/34857 discloses transdermal drug delivery of various active agents using a power density of less than 20 W/cm2, preferably less than 10 W/cm2; the frequency used being less than 2.5 MHz, preferably less than 2 MHz, preferably less than 1 MHz, most preferably 20-100 kHz; Experimental data in vivo on rats was generated using a frequency of 20 kHz and a power density of 1 or 1.5 or 7 W/cm2.
U.S. Pat. No. 4,767,402, describes transdermal drug delivery using ultrasound at a power density of 0-3 W/cm2, preferably 0.5-1.5 MHz, and recommends that as the power density is reduced, the frequency should also be reduced. A power density of 1-2 W/cm2 at frequency 870 kHz is exemplified.
Cosmetic treatments that aim to improve skin quality are also hindered by the barrier function of the epidermis and in particular the outer stratum corneum. The epidermis provides a significant mechanical and chemical barrier to solute transfer due to the cornified cell/lipid bilayer. Also, there is significant enzymatic activity in the epidermis and dermis, which provides a biochemical defence to neutralise applied xenobiotics and which is comparable to that of the liver in terms of activity per unit volume. Additionally, the molecular weight of active substances is known to be important in determining their propensity to diffuse across the skin. Diffusion of substances of molecular weight around 500 Da and above is known to be inefficient. Methods and apparatus involving ultrasound have been described for use in cosmetic of the skin and in medical treatments.
U.S. Pat. No. 6,113,559 discloses a method and apparatus of reducing wrinkles by application of a focused ultrasound beam (ultrasound power density 100-500 W/cm2, frequency 1-500 MHz) to a region of skin, so that the energy delivered to the dermis layer is sufficient to heat the tissue in order to stimulate or irritate the dermis layer, causing a change in the dermis layer that confers a change in smoothness of the epidermis layer.
Ultrasound therapy for the treatment of cellulite is well known and the application of ultrasonic wave energy has generally proven effective in breaking down subcutaneous fatty tissue. As an example, EP 0 695 559, relates to multifunctional equipment for treatments of cellulite, which can include emitters of ultrasonic vibrations for application to, for example, the thighs of a patient's body. However, suitable power densities and frequencies are not discussed. GB 2303552 discloses ultrasound apparatus useful for the non-invasive reduction of cellulite. The ultrasound devices are used for the ultrasonic treatment of cellulite at a predetermined frequency of about 3.3 MHz and a typical power density of 2.8 W/cm2, with 50% of the energy being absorbed within a depth of from 1.27 cm to 2.54 cm below the skin surface.
U.S. Pat. No. 6,030,374 discloses a method for enhancing transport of an active agent through the skin by exposing skin to ultrasound and applying an active agent to the skin by injection. The active agent may be used to reduce the appearance of cellulite. For lower frequency ultrasound, an ultrasound frequency between 25 kHz and 3 MHz at a power density of 0.5-2.0 W/cm2 is used; for higher frequency ultrasound, an ultrasound frequency between 3 MHz and 16 MHz at a power density of 0.2-1.0 W/cm2 is used.
U.S. Pat. No. 5,665,053 relates to an endermology body massager having ultrasound generators that are selectively controlled by the operator. The very low frequency long wave ultrasound disclosed, 10 to 40 kHz, is in the range generally recognised as being disruptive ultrasound, which may be damaging to cells, and thus for safety reasons this is not suitable for general use except at very low power levels.
U.S. Pat. No. 5,507,790 discloses apparatus for focusing ultrasound energy such that the temperature of a site within the patient's subcutaneous adipose tissue layer is raised to between 40.0 and 41.5° C., to accelerate local fat tissue lipolysis reaction rates. The apparatus includes an ultrasonic transducer which supplies ultrasound energy of an undisclosed frequency and at an undisclosed power density to a focusing element.
WO 99/56829 discloses ultrasound bandages and ultrasound transducer array bandages which are said to be useful to accelerate the healing of wounds by positioning the ultrasound bandages and ultrasound transducer array bandages adjacent to a wound and generating ultrasonic pulses.
WO 99/48621 describes large-area flexible piezoelectric composite transducer elements and large-area arrays of such transducer elements have sufficient flexibility to conform to the contours of the human anatomy, e.g., the hip, spine.
To be effective, treatment for cosmetic skin conditions, such as skin ageing and sun damage, must deliver actives to at least the depth of the upper (papillary) dermis and therefore must employ a mechanism to overcome this effective physical and biochemical barrier, even when it has deteriorated with age.
The deterioration of human skin due to natural or ‘intrinsic’ ageing is characterised by a number of symptoms. Such symptoms include a thinning of both the epidermis and the dermis, a flattening of the junction between them, poor wound healing, thermoregulation and immune function along with a deterioration of associated mechanical properties such as tear resistance, elasticity and barrier function. The visible appearance also deteriorates giving a rougher, lined and dry appearance along with uneven pigmentation. In most cases skin ageing is of little medical importance except in such cases as impaired wound healing which allows infection and dysfunction.
Visible deterioration in skin with age is due to a combination of several changes which happen more or less concurrently. This deterioration can be accelerated by lifestyle choices such as smoking and sunbathing. The visibly apparent changes include: sagging skin, rough skin texture, dyspigmentation, dull complexion and a general loss of radiance. Wrinkling, or rhytide formation, is probably the symptom most commonly associated with skin ageing and is known to be caused by a change in the type and distribution of matrix proteins and proteoglycans. Similarly, functions of the skin that decline with age include: cell replacement, immune recognition, sensory perception, injury response, vascular responsiveness, vitamin D production, barrier function, thermoregulation, sebum production, chemical clearance, sweat production and mechanical protection. There may also be changes in pH (from 4.5 to 5).
Ageing skin is characterised by decreased epidermal thickness and proliferation along with the flattening of the rete ridge pattern. The apparent thinning may be linked to increased apoptosis in the basal and spinous layers, in conjunction with impaired cell proliferation of the basal layer. Senescent skin thins, becomes less elastic and has reduced barrier function. This is because the dermis contains a reduced cellular content with stiff, inflexible matrix proteins and a diminished number of capillary loops. The overlying epidermis consequently suffers because the dermal-epidermal junction (DEJ) flattens, resulting in a reduced contact surface area as there are fewer capillary loops in proximity to the DEJ. The exchange of nutrients and metabolites between the two layers decreases and the communication needed to maintain layer integrity in response to changes in external environment conditions is impaired.
The skin is not only subjected to intrinsic or chronological ageing processes, but also environmental or extrinsic ones. For example, factors such as diet, pollution and smoking are known to affect the rate of skin ageing. However one factor stands out as the most potent ‘gerontogen’: sunlight. It has been suggested that approximately 80% of facial ageing is due to sun exposure.
Collagen, elastin and other intra- and extracellular proteins of the skin are affected resulting in solar elastosis, the build-up of localised elastic tissue in fibrous bundles throughout the dermis.
The UV component of sunlight has also been linked to the reduction in cellular population of the epidermis (keratinocytes) and dermis (fibroblasts). It has been suggested that this is due to the increase in programmed cell death or apoptosis. The epidermis and the dermis are known to become increasingly acellular with age, which supports this hypothesis. Despite the epidermis influencing the dry and rough appearance of the skin, it is the dermis that dictates the degree of surface smoothness. Reduction and/or a redistribution of matrix proteins and high water-binding proteoglycans largely govern the appearance of wrinkles and general surface smoothness. Similarly, scarring of the skin is due to abnormal protein content, conformation and distribution via the formation of granulation tissue following trauma, again primarily a dermal rather than an epidermal problem.
Typical symptoms of photoageing include coarseness, wrinkling, irregular pigmentation, telangiectasia, scaliness and a variety of benign, premalignant and malignant neoplasms. Photoageing is predominant in fair-skinned Caucasians who have a history of sun-exposure and occurs most severely on the face, neck and extensor surfaces of the upper extremities. Elastosis, recognised as the pebbly goose flesh seen on the neck and upper chest, is due to nodular aggregations of altered elastin fibres in the dermis. A proliferation of increasingly thickened and tangled elastin fibres has been observed in the papillary and reticular dermis of sun-exposed skin. Even in mildly sun-damaged skin, a 5-20 fold increase in elastin fibre diameter has been found, with slight changes in the fibrillar structure and an alteration of the normal architecture, giving a disrupted and “moth-eaten” appearance.
Overall, photodamage is manifested by the progressive injury to dermal fibroblasts with quantitative and qualitative alterations to the supporting extracellular matrix. As solar energy passes through the skin and is absorbed a gradient of damage occurs, the most damage being seen in the outer papillary dermis, with less to the deeper reticular dermis.
Intrinsic (chronological) aging is characterised by atrophy of skin with loss of elasticity and reduced metabolic activity. Specifically, the stratum corneum remains unchanged, but the epidermis thins overall, with a flattening of the dermal-epidermal junction resulting in increased fragility of the skin. Dermal thickness and dermal vascularity are decreased; this is accompanied by a decrease in the number and the biosynthetic activity of dermal fibroblasts. This latter change is manifested by delayed wound healing. Increasing age also has the effect of reducing the response of keratinocytes and fibroblasts to growth factors.
At the molecular and ultrastructural level, there are changes in elasticity and other changes in matrix proteins. As regards elasticity, there is a reduction in the extracellular protein fibrillin which is a major component of microfibril bundles that connect the dermal-epidermal junction to the papillary dermis. These bundles, often called oxytalan fibres, essentially provide an elastic connection between the epidermis and dermis. Previously considered to be synthesised only by fibroblasts, the fibres present at the dermal-epidermal junction have been shown to be synthesised by keratinocytes. The concentration of fibrillin in photoaged skin has been found to be decreased and has proved to be a useful biomarker for photoageing as it is known to be connected with wrinkle formation. Fibrillin concentration is also reduced in skin that has been subjected to tensile stress and exhibits stretch marks (striae distensae).
In vivo proteins are post-translationally modified by a non-enzymatic reaction (Maillard reaction) between proteins (both intra- and extracellularly) and sugars. This reaction is known either as glycation, or glycosylation, and is well recognized to play an important part in protein turnover, tissue remodelling, diabetes and ageing. In skin, this process is exacerbated by UV, with dermal glycation often increasing significantly after 35 years. Glycation of proteins occurs when reducing sugars such as glucose and fructose, or their reactive intermediates such as glyoxal, react with the amino groups of long half-life proteins such as collagen (t1/2=15 years in human skin) and elastin in the dermis. As a result of this process, cytotoxic Advanced Glycation End-products (AGEs) (AGEs) accumulate.
An increase in glycation has been seen in skin previously irradiated with UV. A well-known biomarker for protein glycation, carboxymethyllysine (CML), has been shown to be present predominantly in areas of solar elastosis in the dermis and generally at higher concentrations in photoaged skin, suggesting that UV-induced oxidation may accelerate the formation AGEs in photoaged skin.
The build-up of AGEs has several effects. Advanced glycation end product-modified proteins are endogenous sensitizers of photo-oxidative cell damage in human skin by UVA-induced generation of reactive oxygen species (ROS) contributing to photoageing and photocarcinogenesis. ROS generation has also been linked to early and late stages of AGE formation with a direct link with the rate of ROS generation which in turn increases matrix metalloproteinase expression with a consequent decrease in healthy digestible matrix. There is also cross-linking of extra-cellular proteins which causes deterioration of the structural mechanical properties of the protein and reduces their susceptibility to the body's natural enzymes, such as matrix metalloproteinases (MMPs), which normally ensure a regular, healthy protein turnover. Cross-linking AGEs include species such as pentosidine. Non-cross-linking AGEs include species such as CML. Glycation also decreases water accessibility of proteins making them more heat stable and less likely to be thermally denatured.
The body has a host of physiological mechanisms that defend against deleterious protein modifications, including protein-digesting enzymes. Timely proteolysis removes damaged proteins before they undergo oxidative damage and cross-linking. Therefore, rapid effective proteolysis is essentially an anti-aging mechanism. It has been mentioned already that proteins such as collagen and elastin, which have been post-translationally modified through UV-induced glycation, are more resistant to digestion by endogenous enzymes (e.g. metalloproteinases). This, coupled with the increase in expression of such enzymes, further reduces the ratio of healthy digestible matrix proteins to modified deleterious proteins.
Not only are native proteins turned over by endogenous enzymes such as collagenase and elastase, but other systems are present both intra- and extracellularly to deal with ageing and/or denatured/stressed proteins. One such mechanism employs molecular chaperones. Increasing age is associated with a reduced capacity to maintain homeostasis in all physiological systems and this may result, in part at least, from a parallel and progressive decline in the ability to produce heat shock proteins. An attenuated heat shock protein response may contribute to increased susceptibility to environmental challenges in aged individuals.
Heat Shock Proteins (HSPs), also known as stress proteins, are thought to act as molecular chaperones by assisting with protein synthesis, transport, folding and degradation. They are a group of proteins that are present in all cells, in all life forms. They are induced when a cell undergoes environmental stress, heat, cold, or oxygen deprivation. HSPs are also present in cells under perfectly normal conditions and have been linked to modulation of contraction and relaxation responses in vascular smooth muscle; they play an important role in protein folding and function, even in the absence of stress.
The formation of Advanced Glycation End-products causes protein unfolding irreversible cross-linking and other chemical modifications. HSPs are known to promote refolding/maintenance of conformation and also the rapid degradation of irreversibly-damaged proteins. Small heat shock proteins, such as α-crystallin, are known to protect eye lens proteins from glycation induced changes. Small heat shock proteins (sHSPs) are known to have common ‘crystallin’ core that appears to be responsible for the catalytic activity of these chaperones. It has been suggested that a greater understanding of α-crystallin/sHsp chaperone action will have implications for the development of therapeutics to treat and prevent cataract.
The heat shock protein family includes the 8-kD ubiquitin (known in connection with the ubiquitin-proteasome protein degradation pathway), 32-kD heme oxygenase-1 (connected to UVA induced oxidative stress) and HSP-47, a known collagen chaperone. HSP-27 has been found in human skin and has been suggested to play a protective role in inflammatory diseases due to its links with interleukin-1 and tumour necrosis factor-α. This, along with the understanding that HSP-27 expression is closely linked with epidermal keratinocyte differentiation suggests that heat shock proteins such as HSP-27 play a role in skin protection and possibly in the UV-sunburn inflammation cycle. In contrast to other cells and organ systems, epidermal keratinocytes are known to express HSP-72 constitutively, i.e. without exposure to previous stress. The heat shock protein HSP47 has been shown to be important as a molecular chaperone for procollagen synthesis in human fibroblasts. HSP47 synthesis is reduced in aged and photo-aged skin.
HSP expression following exposure to UV has been linked with increased resistance to UV-induced cell death. Non-toxic inducers of HSPs may protect against the immediate and long-term effects of UV exposure. Studies have shown that prior exposure of cells to red and infra-red (IR) light protects them against subsequent exposure to UV light. Similarly, IR pre-treatment of cells also protects cells against subsequent lethal (51° C.) applied heat stress.
The well-known protective effect of HSPs from environmental stress is not constant with age. The HSP response to stress is attenuated with age, probably at the transcriptional level. Repetitive mild heat shock (RMHS) of human skin fibroblasts has been found to reduce the rate of age-related changes. One study has connected the age-related decrease in the ability of human fibroblasts to reduce the accumulation of glycated proteins with a parallel reduction in the ability to express HSP70, as human fibroblasts exposed to RMHS exhibited increased HSP70 expression and reduced accumulation of glycated protein accumulation. The beneficial effects of RMHS have been attributed to increased proteasomal activity, increased ability to decompose H2O2, reduced accumulation of lipofuscin and an enhanced resistance to UVA radiation.
Temperature rises of 3-5° C. above baseline in muscle have been shown to cause the induction of HSPs. Induction of HSPs by 30 mins of pulsed ultrasound applied at normal body temperature has been demonstrated in the rat embryo, showing that the heat shock response is not specific to heat but can occur in response to mechanical stress. Similarly, chick embryos exposed to ultrasound, without any significant thermal contribution, have shown heightened synthesis of HSP72 suggesting that the mechanical stimulus can induce a stress response. It was also concluded that to produce a ‘full biological effect, stress must be constant for approximately 10 s or more over any time interval during exposure’. It is possible that cumulative effects can stimulate HSP production as has been found when mild heat shock was repeated over 3 days causing significantly elevated muscle HSP levels.
Certain substances have an effect on HSP expression. For example, salicin has been shown to reduce the necessary degree of temperature rise from 42° C. to 39° C. to elicit HSP expression and to reduce the degree of subsequent UV-induced damage in cultured human fibroblasts and keratinocytes. Known irritants such sodium lauryl sulphate (SLS) also induce HSP expression. HSP27 upregulation due to SLS application to excised human skin has been used as a method of determining cellular stress due to chemical irritancy. In a similar study, however, SLS induced expression of HSP27 in human epidermis was suppressed by topical application of vitamin C.
The substance zinc-L-carnosine, known also as Polaprezinc commercially, has been shown to induce HSP72 (stress-induced HSP70) expression in gastric mucosa protecting cells from applied stress through chemical irritancy. As a control, ZnSO4 and carnosine were also tested and found not to elicit the same response. Known as an anti-ulcer drug, zinc-L-carnosine's wound-healing action has been linked to its proliferative response in non-endothelial cells such as fibroblasts.
The influence of aspirin on HSP70 expression in intact rats subjected to heat stress has been investigated. Rats were injected intraperitoneally either with aspirin (100 mg/kg), or vehicle alone, 60 min prior to their placement at 37° C. or room temperature for 30 min. The combination of aspirin with heat treatment resulted in 3 to 4 fold higher levels of HSP70 mRNA relative to those seen with heat treatment alone.
The role of HSP-72 and -70 in conferring resistance to aspirin attack of the rat gastric mucosa has been investigated; expression of these HSPs was elevated following chronic exposure to aspirin.
Analgesics such as aspirin, ibuprofen and paracetamol are known to protect against cataract. This action has been attributed to the inhibition of sugar-induced cross-linking in small HSPs such as α-crystallin. Enzymes that protect against cataract are prone to glycation-induced inactivation, but aspirin has been shown to protect against this.
Similarly, acetyl-L-carnitine has been recognised as a potential chaperone-protecting agent due to its abilities to acetylate potential glycation sites of small HSPs and correspondingly protect them from glycation-mediated protein damage.
Small heat shock proteins (sHSPs) and Clusterin are molecular chaperones that share many functional similarities despite their lack of significant sequence similarity. Small heat shock proteins are ubiquitous intracellular proteins whereas clusterin is generally found extracellularly. Both chaperones prevent the amorphous aggregation and precipitation of target proteins under stress conditions such as elevated temperature, reduction and oxidation. Transcription of both HSPs and clusterin are mediated by the transcription factor HSF-1. However, clusterin has been shown to be much more efficient than certain sHSPs, such as α-crystallin, in preventing the precipitation from solution of stressed target proteins.
Clusterin is expressed as a 75-80 kDa heterodimeric protein that is heavily glycated such that 30% of its mass is comprised of sugar. Whereas the chaperone activity of small heat shock proteins such as α-crystallin is reduced significantly at lower pH, the activity of clusterin is enhanced at lower pH. This has important implications for sites of tissue damage or inflammation where local acidosis (pH<6) occurs. Another similarity that clusterin shares with sHSPs is the ability to regulate apoptosis. Over-expression of clusterin can protect cells from a variety of agents (e.g. TNF-α and UV irradiation) that otherwise induce apoptosis. It has been suggested that clusterin may interact with stressed cell surface proteins to inhibit pro-apoptotic signal transduction or prevent inappropriate interactions of intracellular proteins during stress.
Many topical skin preparations are available for the treatment of medical skin conditions and for the treatment of cosmetic skin conditions, in particular skin ageing and sun damage. In many instances these preparations are ineffective, with only minimal or short lived efficacy. There is thus a desire for new preparations effective in the treatment of skin conditions. Furthermore, the present invention addresses the problems of achieving efficient delivery to the skin of such novel preparations.